Download the complete set of whole mitochondrial genomes into the directory called “mitochondria”. Note that MITOCHONDRIA was archived. You are about to download the mitochondrial genomes from 7681 species and you don’t have to click 7681 times! But it will take a while. Make sure the internet connection is good through out the period. I started downloading before I went to bed, and when I woke up, there were there in the folder of mitochondria.
mkdir mitochondria
cd mitochondria
wget 'ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_refseq/MITOCHONDRIA/Metazoa/*.gbk'
cd ..Download the NCBI taxonomy, which contains all the relationship between taxa. Each taxon is identified by a unique numerical id: taxid. You can check the content of files, e.g. show the first 6 lines of the file. Then reformat it according to the OBITools format.
mkdir ncbitaxon
cd ncbitaxon/
wget 'ftp://ftp.ncbi.nlm.nih.gov://pub/taxonomy/taxdump.tar.gz'
tar -xf taxdump.tar.gz
head -6 nodes.dmp
cd ..obitaxonomy -t ncbitaxon -d ncbitaxon
ls
## WolfDietMetabarcoding.Rproj
## amniota.ecoprimers
## ecoPCR
## ecoPrimers
## mito.all.fasta
## mito.one.fasta
## mito.vert.adx
## mito.vert.fasta
## mito.vert.ndx
## mito.vert.rdx
## mito.vert.tdx
## mito.vert_001.sdx
## ncbitaxon
## ncbitaxon.adx
## ncbitaxon.ndx
## ncbitaxon.rdx
## ncbitaxon.tdx
## readme.md
## vert.01.ecopcr
## vert.02.ecopcr
## vert.03.ecopcr
## vert.04.ecopcr
## vert.05.ecopcr
## vert.06.ecopcr
## vert.07.ecopcr
## vert.Riaz.ecopcr
## vertebrata.ecoprimers
## wolf.md
## wolf.rmd
## wolf_cache
## wolf_files
## wolfhtml.html
## wolfhtml.rmd
As you can tell, there are four new files generated, .adx, .ndx, .rdx and .tdx.
obiconvert mitochondria/*.gbk > mito.all.fasta
A new file called “mito.all.fasta” is generated which contains complete mitochondrial genomes from all the species.
head -5 mito.all.fasta
## >AC_000022 organism=Rattus norvegicus; taxid=10116; Rattus norvegicus strain Wistar mitochondrion, complete genome
## gttaatgtagcttataataaagcaaagcactgaaaatgcttagatggattcaaaaatccc
## ataaacacaaaggtttggtcctggccttataattaattggaggtaagattacacatgcaa
## acatccataaaccggtgtaaaatcccttaaagatttgcctaaaacttaaggagagggcat
## caagcacataatatagctcaagacgccttgcctagccacacccccacgggactcagcagt
taxid, and keep a single genome per speciesNote: obiannotate command adds/edits sequence record annotations.
obiannotate -d ncbitaxon --with-taxon-at-rank=species mito.all.fasta |
obiannotate -S 'ori_taxid=taxid' |
obiannotate -S 'taxid=species' |
obiuniq -c taxid |
obiselect -c taxid -n 1 -f count -M > mito.one.fasta
obicount mito.all.fasta
obicount mito.one.fasta
In mito.all.fasta, there are 5669 genomes, whereas in mito.one.fasta, there are 5651 genomes. It shows there are multiple complete mitochondrial genomes for certain species.
Selection is done with taxid, so first we will need to find the taxid for the phylum vertebrata.
ecoPCR/src/ecofind -d ncbitaxon '^vertebrata$'
## Reading 1739138 taxa...
## No local taxon
## #
## # opening ncbitaxon database
## # 1739138 taxons
## #
## # searching for '^vertebrata$' pattern
## # taxonomy id | taxonomy rank | name | class name | scientific name
## #
## 7742 | no rank | Vertebrata | scientific name | Vertebrata
## 1261581 | genus | Vertebrata | scientific name | Vertebrata
## # 2 records found
So there are two records called “vertebrata”. In fact, there is a genus called vertebrata. Which one is what we want?
ecoPCR/src/ecofind -d ncbitaxon -p 7742
ecoPCR/src/ecofind -d ncbitaxon -p 1261581
## Reading 1739138 taxa...
## No local taxon
## #
## # opening ncbitaxon database
## # taxonomy id | taxonomy rank | name | class name | scientific name
## #
## 7742 | no rank | Vertebrata | scientific name | Vertebrata
## 89593 | subphylum | Craniata | scientific name | Craniata
## 7711 | phylum | Chordata | scientific name | Chordata
## 33511 | no rank | Deuterostomia | scientific name | Deuterostomia
## 33213 | no rank | Bilateria | scientific name | Bilateria
## 6072 | no rank | Eumetazoa | scientific name | Eumetazoa
## 33208 | kingdom | Metazoa | scientific name | Metazoa
## 33154 | no rank | Opisthokonta | scientific name | Opisthokonta
## 2759 | superkingdom | Eukaryota | scientific name | Eukaryota
## 131567 | no rank | cellular organisms | scientific name | cellular organisms
## 1 | no rank | root | scientific name | root
## # 10 parent(s) found
## #
## Reading 1739138 taxa...
## No local taxon
## #
## # opening ncbitaxon database
## # taxonomy id | taxonomy rank | name | class name | scientific name
## #
## 1261581 | genus | Vertebrata | scientific name | Vertebrata
## 2008651 | subfamily | Polysiphonioideae | scientific name | Polysiphonioideae
## 2803 | family | Rhodomelaceae | scientific name | Rhodomelaceae
## 2802 | order | Ceramiales | scientific name | Ceramiales
## 2045261 | subclass | Rhodymeniophycidae | scientific name | Rhodymeniophycidae
## 2806 | class | Florideophyceae | scientific name | Florideophyceae
## 2763 | no rank | Rhodophyta | scientific name | Rhodophyta
## 2759 | superkingdom | Eukaryota | scientific name | Eukaryota
## 131567 | no rank | cellular organisms | scientific name | cellular organisms
## 1 | no rank | root | scientific name | root
## # 9 parent(s) found
## #
7742 is what we want for the phylum Vertebrata. Use taxid 7742 to select only vertebrata genomes.
obigrep -d ncbitaxon -r 7742 mito.one.fasta > mito.vert.fasta
obicount mito.vert.fasta
The newly generated mito.vert.fasta has 3717 records of sequences.
Reformat the vertebrata fasta file into an ecoPCR database.
obiconvert -d ncbitaxon --ecopcrdb-output=mito.vert mito.vert.fasta
Four new files are generated, mito.vert.rdx, .tdx, .ndx, and .adx.
ecoPrimers/src/ecoPrimers -h
ecoPCR/src/ecoPCR -h
We want to focus on just mammals and birds. What is the most recent ancestor for mammals and birds? Amniota! Amniota is a clade of tetrapod vertebrates comprising the reptiles, birds and mammals. Amniotes lay their eggs on land or retain the fertimized egg within the mother, and is distinguished from the anamniotes (fish and amphibians), which typically lay their eggs in water.
ecoPCR/src/ecofind -d ncbitaxon '^amniota$'
## Reading 1739138 taxa...
## No local taxon
## #
## # opening ncbitaxon database
## # 1739138 taxons
## #
## # searching for '^amniota$' pattern
## # taxonomy id | taxonomy rank | name | class name | scientific name
## #
## 32524 | no rank | Amniota | scientific name | Amniota
## # 1 records found
The taxid for Amniota is 32524.
ecoPCR/src/ecofind -d ncbitaxon -p 32524
## Reading 1739138 taxa...
## No local taxon
## #
## # opening ncbitaxon database
## # taxonomy id | taxonomy rank | name | class name | scientific name
## #
## 32524 | no rank | Amniota | scientific name | Amniota
## 32523 | no rank | Tetrapoda | scientific name | Tetrapoda
## 1338369 | no rank | Dipnotetrapodomorpha | scientific name | Dipnotetrapodomorpha
## 8287 | superclass | Sarcopterygii | scientific name | Sarcopterygii
## 117571 | no rank | Euteleostomi | scientific name | Euteleostomi
## 117570 | no rank | Teleostomi | scientific name | Teleostomi
## 7776 | no rank | Gnathostomata | scientific name | Gnathostomata
## 7742 | no rank | Vertebrata | scientific name | Vertebrata
## 89593 | subphylum | Craniata | scientific name | Craniata
## 7711 | phylum | Chordata | scientific name | Chordata
## 33511 | no rank | Deuterostomia | scientific name | Deuterostomia
## 33213 | no rank | Bilateria | scientific name | Bilateria
## 6072 | no rank | Eumetazoa | scientific name | Eumetazoa
## 33208 | kingdom | Metazoa | scientific name | Metazoa
## 33154 | no rank | Opisthokonta | scientific name | Opisthokonta
## 2759 | superkingdom | Eukaryota | scientific name | Eukaryota
## 131567 | no rank | cellular organisms | scientific name | cellular organisms
## 1 | no rank | root | scientific name | root
## # 17 parent(s) found
## #
Design a metabarcode (50-120 bp long) for Vertebrata, with maximum mismatch rate (-e) as 3, restrict (-r) to Vertebrata (taxid:7742) to optimize primers for Vertebrata.
ecoPrimers/src/ecoPrimers -d mito.vert -e 3 -l 50 -L 120 -r 7742 -c > vertebrata.ecoprimers
Yeah!!! ecoPrimers found 7 good pairs. Now do the same thing for Amniota, restrict (-r) to Amniota (taxid:32524) to optimize primers for Amniota.
ecoPrimers/src/ecoPrimers -d mito.vert -e 3 -l 50 -L 120 -r 7742 -c > amniota.ecoprimers
ecoPrimers couldn’t find any good primer pair:( Okay, let’s test the 7 primer pairs we found which are vertebrate universal.
cat vertebrata.ecoprimers
## #
## # ecoPrimer version 0.4
## # Rank level optimisation : species
## # max error count by oligonucleotide : 3
## #
## # Restricted to taxon:
## # 7742 : Vertebrata (no rank)
## #
## # strict primer quorum : 0.70
## # example quorum : 0.90
## # counterexample quorum : 0.10
## #
## # database : mito.vert
## # Database is constituted of 3717 examples corresponding to 3646 species
## # and 0 counterexamples corresponding to 0 species
## #
## # amplifiat length between [50,120] bp
## # DB sequences are considered as circular
## # Pairs having specificity less than 0.60 will be ignored
## #
## 0 AAACTGGGATTAGATACC TAGAACAGGCTCCTCTAG 48.5 14.9 52.3 23.1 7 9 GG 3675 0 0.989 3605 0 0.989 2653 0.736 82 119 107.70
## 1 AACTGGGATTAGATACCC TAGAACAGGCTCCTCTAG 50.5 22.7 52.3 23.1 8 9 GG 3675 0 0.989 3605 0 0.989 2653 0.736 81 118 106.70
## 2 CAAACTGGGATTAGATAC TAGAACAGGCTCCTCTAG 47.5 26.4 52.3 23.1 7 9 GG 3672 0 0.988 3602 0 0.988 2650 0.736 83 119 108.70
## 3 ATTAGATACCCCACTATG TAGAACAGGCTCCTCTAG 47.6 10.9 52.3 23.1 7 9 GG 3669 0 0.987 3599 0 0.987 2643 0.734 74 111 99.70
## 4 GATTAGATACCCCACTAT TAGAACAGGCTCCTCTAG 47.2 10.3 52.3 23.1 7 9 GG 3668 0 0.987 3598 0 0.987 2642 0.734 62 112 100.69
## 5 ACTGGGATTAGATACCCC TAGAACAGGCTCCTCTAG 52.6 22.1 52.3 23.1 9 9 GG 3634 0 0.978 3567 0 0.978 2615 0.733 85 117 105.76
## 6 GGGATTAGATACCCCACT TAGAACAGGCTCCTCTAG 52.6 21.3 52.3 23.1 9 9 GG 3633 0 0.977 3566 0 0.978 2610 0.732 82 114 102.75
ecoPCRThe seven primer pairs are annotated as No.01 - No.07 and Riaz cited from Riaz et al. 2011, as shown in the output table.
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c AAACTGGGATTAGATACC TAGAACAGGCTCCTCTAG > vert.01.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c AACTGGGATTAGATACCC TAGAACAGGCTCCTCTAG > vert.02.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c CAAACTGGGATTAGATAC TAGAACAGGCTCCTCTAG > vert.03.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c ATTAGATACCCCACTATG TAGAACAGGCTCCTCTAG > vert.04.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c GATTAGATACCCCACTAT TAGAACAGGCTCCTCTAG > vert.05.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c ACTGGGATTAGATACCCC TAGAACAGGCTCCTCTAG > vert.06.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c GGGATTAGATACCCCACT TAGAACAGGCTCCTCTAG > vert.07.ecopcr
ecoPCR/src/ecoPCR -d mito.vert -e 3 -l 50 -L 120 -c TTAGATACCCCACTATGC TAGAACAGGCTCCTCTAG > vert.Riaz.ecopcr
Download and install the R packages: ROBITools, ROBITaxonomy, ROBIBarcodes. See more.
Import libraries and the data
library(ROBITools)
library(ROBITaxonomy)
library(ROBIBarcodes)
taxo=read.taxonomy("ncbitaxon")
Find taxID for important groups
vert.id=ecofind(taxo,'^vertebrata$')[1]
mam.id=ecofind(taxo,'^mammalia$')
ave.id=ecofind(taxo,'^aves$')
rum.id=ecofind(taxo,'^ruminantia$')
carn.id=ecofind(taxo,'^carnivora$')
rod.id=ecofind(taxo,'^rodentia$')
lago.id=ecofind(taxo,'^lagomorpha$')
ins.id=ecofind(taxo,'^insectivora$')
Import the ecoPCR results for primer pair No.01.
vert01=read.ecopcr.result("vert.01.ecopcr")
is_a_vert=is.subcladeof(taxo,vert01$taxid,vert.id)
table(is_a_vert)
## is_a_vert
## TRUE
## 3680
is_a_mam=is.subcladeof(taxo,vert01$taxid,mam.id)
table(is_a_mam)
## is_a_mam
## FALSE TRUE
## 2887 793
is_a_ave=is.subcladeof(taxo,vert01$taxid,ave.id)
table(is_a_ave)
## is_a_ave
## FALSE TRUE
## 3201 479
is_a_rum=is.subcladeof(taxo,vert01$taxid,rum.id)
table(is_a_rum)
## is_a_rum
## FALSE TRUE
## 3510 170
is_a_carn=is.subcladeof(taxo,vert01$taxid,carn.id)
table(is_a_carn)
## is_a_carn
## FALSE TRUE
## 3548 132
is_a_rod=is.subcladeof(taxo,vert01$taxid,rod.id)
table(is_a_rod)
## is_a_rod
## FALSE TRUE
## 3572 108
is_a_lago=is.subcladeof(taxo,vert01$taxid,lago.id)
table(is_a_lago)
## is_a_lago
## FALSE TRUE
## 3666 14
is_a_ins=is.subcladeof(taxo,vert01$taxid,ins.id)
table(is_a_ins)
## is_a_ins
## FALSE TRUE
## 3645 35
vert.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_vert)
vert.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_vert)
mam.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_mam)
mam.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_mam)
ave.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_ave)
ave.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_ave)
rum.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_rum)
rum.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_rum)
carn.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_carn)
carn.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_carn)
rod.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_rod)
rod.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_rod)
lago.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_lago)
lago.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_lago)
ins.01.forward=ecopcr.forward.shanon(ecopcr=vert01, group=is_a_ins)
ins.01.reverse=ecopcr.reverse.shanon(ecopcr=vert01, group=is_a_ins)
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
par(mfcol=c(3,2), mar=c(3,1,2,1))
dnalogoplot(vert.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Vertebrata")
dnalogoplot(mam.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Mammalia")
dnalogoplot(ave.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Aves")
dnalogoplot(vert.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Vertebrata")
dnalogoplot(mam.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reerse Mammalia")
dnalogoplot(ave.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Aves")
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
par(mfcol=c(5,2), mar=c(2,1,1,1))
dnalogoplot(rum.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Ruminantia")
dnalogoplot(carn.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Carnivora")
dnalogoplot(rod.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Rodentia")
dnalogoplot(lago.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Lagomorpha")
dnalogoplot(ins.01.forward$"TRUE",
primer="AAACTGGGATTAGATACC",
main="Forward Insectivora")
dnalogoplot(rum.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Ruminantia")
dnalogoplot(carn.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Carnivora")
dnalogoplot(rod.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Rodentia")
dnalogoplot(lago.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Lagomorpha")
dnalogoplot(ins.01.reverse$"TRUE",
primer="TAGAACAGGCTCCTCTAG",
main="Reverse Insectivora")
par(mfcol=c(1,1))
mismatchplot(vert01)
Vertebrata:
only.vert=vert01[is_a_vert,]
res = resolution(taxo,only.vert)
resolution = with(only.vert,
unique(data.frame(species_name,taxid,rank=res))
)
t(t(sort(table(resolution$rank)/length(resolution$rank),decreasing = TRUE)))
##
## [,1]
## species 0.7238095238
## genus 0.1338775510
## family 0.0778231293
## subfamily 0.0282993197
## order 0.0127891156
## subspecies 0.0087074830
## no rank 0.0051700680
## subgenus 0.0035374150
## tribe 0.0027210884
## infraorder 0.0013605442
## superfamily 0.0013605442
## superorder 0.0005442177
Mammalia:
##
## [,1]
## species 0.75662043
## genus 0.16141236
## family 0.04035309
## subfamily 0.02395965
## subspecies 0.01134931
## subgenus 0.00630517
Aves:
##
## [,1]
## species 0.638830898
## family 0.104384134
## genus 0.087682672
## order 0.087682672
## subfamily 0.054279749
## subspecies 0.012526096
## superfamily 0.010438413
## superorder 0.004175365
for_tm=data.frame("tm"=vert01$forward_tm,"direction"=rep("forward",length(vert01$forward_tm)))
rev_tm=data.frame("tm"=vert01$reverse_tm,"direction"=rep("reverse",length(vert01$reverse_tm)))
tm.df=rbind(for_tm,rev_tm)
boxplot(tm~direction,tm.df, ylab="Tm")
Import the ecoPCR results for primer pair No.02.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.7238095238
## genus 0.1338775510
## family 0.0778231293
## subfamily 0.0282993197
## order 0.0127891156
## subspecies 0.0087074830
## no rank 0.0051700680
## subgenus 0.0035374150
## tribe 0.0027210884
## infraorder 0.0013605442
## superfamily 0.0013605442
## superorder 0.0005442177
Mammalia:
##
## [,1]
## species 0.75662043
## genus 0.16141236
## family 0.04035309
## subfamily 0.02395965
## subspecies 0.01134931
## subgenus 0.00630517
Aves:
##
## [,1]
## species 0.638830898
## family 0.104384134
## genus 0.087682672
## order 0.087682672
## subfamily 0.054279749
## subspecies 0.012526096
## superfamily 0.010438413
## superorder 0.004175365
Import the ecoPCR results for primer pair No.03.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.723659134
## genus 0.133950449
## family 0.077865505
## subfamily 0.028314729
## order 0.012796079
## subspecies 0.008712224
## no rank 0.005172883
## subgenus 0.003539341
## tribe 0.002722570
## infraorder 0.001361285
## superfamily 0.001361285
## superorder 0.000544514
Mammalia:
##
## [,1]
## species 0.75662043
## genus 0.16141236
## family 0.04035309
## subfamily 0.02395965
## subspecies 0.01134931
## subgenus 0.00630517
Aves:
##
## [,1]
## species 0.63807531
## family 0.10460251
## genus 0.08786611
## order 0.08786611
## subfamily 0.05439331
## subspecies 0.01255230
## superfamily 0.01046025
## superorder 0.00418410
Import the ecoPCR results for primer pair No.04.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.7221919302
## genus 0.1341330425
## family 0.0787895311
## subfamily 0.0286259542
## order 0.0128135224
## subspecies 0.0087241003
## no rank 0.0051799346
## subgenus 0.0035441658
## tribe 0.0027262814
## infraorder 0.0013631407
## superfamily 0.0013631407
## superorder 0.0005452563
Mammalia:
##
## [,1]
## species 0.753787879
## genus 0.164141414
## family 0.040404040
## subfamily 0.023989899
## subspecies 0.011363636
## subgenus 0.006313131
Aves:
##
## [,1]
## species 0.635021097
## family 0.107594937
## order 0.088607595
## genus 0.084388186
## subfamily 0.056962025
## subspecies 0.012658228
## superfamily 0.010548523
## superorder 0.004219409
Import the ecoPCR results for primer pair No.05.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.722116171
## genus 0.134169621
## family 0.078811017
## subfamily 0.028633761
## order 0.012817017
## subspecies 0.008726479
## no rank 0.005181347
## subgenus 0.003545132
## tribe 0.002727025
## infraorder 0.001363512
## superfamily 0.001363512
## superorder 0.000545405
Mammalia:
##
## [,1]
## species 0.753476612
## genus 0.164348925
## family 0.040455120
## subfamily 0.024020228
## subspecies 0.011378003
## subgenus 0.006321113
Aves:
##
## [,1]
## species 0.635021097
## family 0.107594937
## order 0.088607595
## genus 0.084388186
## subfamily 0.056962025
## subspecies 0.012658228
## superfamily 0.010548523
## superorder 0.004219409
Import the ecoPCR results for primer pair No.06.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.7220693451
## genus 0.1351128233
## family 0.0787011558
## subfamily 0.0286186021
## order 0.0129334067
## subspecies 0.0077050083
## no rank 0.0052283985
## subgenus 0.0035773253
## tribe 0.0027517887
## infraorder 0.0013758943
## superfamily 0.0013758943
## superorder 0.0005503577
Mammalia:
##
## [,1]
## species 0.75409836
## genus 0.16393443
## family 0.04035309
## subfamily 0.02395965
## subspecies 0.01134931
## subgenus 0.00630517
Aves:
##
## [,1]
## species 0.638830898
## family 0.104384134
## genus 0.087682672
## order 0.087682672
## subfamily 0.054279749
## subspecies 0.012526096
## superfamily 0.010438413
## superorder 0.004175365
Import the ecoPCR results for primer pair No.07.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.7208149780
## genus 0.1351872247
## family 0.0795704846
## subfamily 0.0289096916
## order 0.0129405286
## subspecies 0.0077092511
## no rank 0.0052312775
## subgenus 0.0035792952
## tribe 0.0027533040
## infraorder 0.0013766520
## superfamily 0.0013766520
## superorder 0.0005506608
Mammalia:
##
## [,1]
## species 0.753787879
## genus 0.164141414
## family 0.040404040
## subfamily 0.023989899
## subspecies 0.011363636
## subgenus 0.006313131
Aves:
##
## [,1]
## species 0.63389121
## family 0.10669456
## genus 0.08786611
## order 0.08786611
## subfamily 0.05648536
## subspecies 0.01255230
## superfamily 0.01046025
## superorder 0.00418410
Import the ecoPCR results for primer pair No.Riaz.
Compare the conservation of the priming sites for Vertebrata, Mammalia and Aves.
Compare the conservation of the priming sites for Ruminantia, Carnivora, Rodentia, Lagomorpha and Insectivora.
Vertebrata:
##
## [,1]
## species 0.7213740458
## genus 0.1344056707
## family 0.0793347874
## subfamily 0.0286259542
## order 0.0128135224
## subspecies 0.0087241003
## no rank 0.0051799346
## subgenus 0.0035441658
## tribe 0.0027262814
## infraorder 0.0013631407
## superfamily 0.0013631407
## superorder 0.0005452563
Mammalia:
##
## [,1]
## species 0.752525253
## genus 0.165404040
## family 0.040404040
## subfamily 0.023989899
## subspecies 0.011363636
## subgenus 0.006313131
Aves:
##
## [,1]
## species 0.630801688
## family 0.111814346
## order 0.088607595
## genus 0.084388186
## subfamily 0.056962025
## subspecies 0.012658228
## superfamily 0.010548523
## superorder 0.004219409
According to conservation of priming sites and taxonomic resolution, primer pair No. 1 and No. 2 are similar and the best. We will order these two primer pairs and try them out!
Forward_No1 AAACTGGGATTAGATACC Amplicon size:107.70 bp (82-119)
Forward_No2 AACTGGGATTAGATACCC Amplicon size:106.70 bp (81-118)
Reverse TAGAACAGGCTCCTCTAG